MACS sorting of TAMs from human and mouse lung tumor tissues

Protocol Title: MACS sorting of TAMs from human lung tumor tissues

1. Prepare single-cell suspension from human lung tumors using the Tumor Dissociation Kit (Order no. 130-095-929, Miltenyi Biotec) according to the manufacturer’s instructions.
2. Perform magnetic sorting of macrophages from single-cell suspensions using primary antibody, human CD68-PE (Order no. 130-118-486, Miltenyi Biotec) and secondary antibody, anti-PE MicroBeads (Order no. 130-048-801, Miltenyi Biotec) according to the instructions provided by the manufacturer.

Protocol Title: MACS sorting of TAMs from mouse lung tumor tissues

1. Fragment mouse tumor tissues (0,5 gm) into small pieces.
2. Incubate in 2mL of RPMI medium with collagenase (5 μg/μl), DNase (10 μg/μl), and 1%P/S for 30 min at 37°C, mixing 300 rpm.
3. Pass cell suspension through 70µM cell strainer.
4. Centrifuge at 500 g, 10 min, 4°C.
5. Pellet down and resuspend the pellet in 5mL of 1X RBC lysis buffer, followed by 15 mins of incubation at RT.
6. Add 10 mL 1X PBS.
7. Centrifuge at 500 g, 10 min, 4°C.
8. Add 10 mL 1X PBS.
9. Centrifuge at 500 g, 10 min, 4°C.
10. Resuspend the pellet in MACS buffer (Miltenyi Biotec) supplemented with 5% BSA and count the number of cells.
11. Perform magnetic sorting of macrophages from single-cell suspension using primary antibody, F4/80-PE mouse (Order no. 130-116-499, Miltenyi Biotec) and secondary antibody, anti-PE MicroBeads (Order no. 130-048-801, Miltenyi Biotec) according to the instructions provided by the manufacturer.